Application Of Biofunctionalized Nanoparticles For Detection Of Escherichia Coli O157:h7

E. coli O157:H7 (Escherichia coli O157:H7, E. coli O157:H7) is the enterohemorrhagic E. coli serotype most often associated with disease outbreaks and with the onset of severe disease in worldwide, which resulted in serious threat for people health and huge economic losses. In this paper, we describe two methods for E. coli O157:H7 object detection based biofunctionalized nanoparticles, and applied methods for the detection of contamination of milk by E. coli O157:H7. The main research areas:Preparation of biofunctionalized nanoparticles. Approximately 13 nm diameter gold nanoparticles (Au-NPs) were prepared by the citrate reduction of HAuCl4. Biofunctionalized Au-NPs are successfully manufactured with Au-NPs, E. coli O157:H7 polyclonal antibody, thiol-capped capture DNA, bio-bar code DNA and DNA probes, and identified through UV-vis, TEM, etc. Magnetic Fe3O4 nanoparticles (MNPs) were synthesized by using chemical coprecipitation technique. The antibodies were immobilized on MNPs by conventional methods using 3-Aminopropyltriethoxysilane (APTES) and subsequent activation by glutaraldehyde.Biofunctionalized nanoparticles for detection of E. coli O157:H7. PCR-based bio-bar codes for detection of E. coli O157:H7. The immune reaction binds to E. coli O157:H7 between the biofunctionalized MNPs and Au-NPs. The sandwich structure MNPs/target/Au-NPs was formed and magnetically separated. After that, dehybridization of the oligonucleotides on the Au-NPs surface allows the determination of the presence of E. coli O157:H7 by identifying the oligonucleotide sequence released from the Au-NPs through PCR. The detection limit of about 10 CFU/mL in pure bacteria sample, and the detection range was 10～106 CFU/mL. For evaluating the specificity, other E. coli serotypes (E. coli O86:H2, O111:K58, K12) and Salmonella Typhimurium were tested as potential competing bacteria of E. coli O157:H7. Results showed that both of other E. coli serotypes and Salmonella Typhimurium did not interfere with the detection of E. coli O157:H7. The intra and inter-assay coefficients of variation were (6.6±3.3)% and (7.3±3.6)% respectively. Based upon the first method, the bio-bar DNA released from the Au-NPs. After that, the bio-bar DNA hybridization to DNA probes results in a color change of Au-NPs. A method established for E. coli O157:H7 based on bio-bar DNA and Au-NPs. The method detection limit was about 102 CFU/mL in pure broth culture, and detection range of 102～106 CFU/mL. Otherwise, the results showed that the assay has a high specificity. The intra-and inter-assay coefficients of variation were (2.9±1.3)%,(3.2±1.5)%, respectively.Detection of E. coli O157:H7 in artificially contaminated milk. PCR-based bio-bar codes for detection of contamination of milk by E. coli O157:H7, detection sensitivity was 10 CFU/mL. Detection of E. coli O157:H7 in artificially contaminated milk based on bio-bar DNA and Au-NPs, and the detection limit was 102 CFU/mL, and results of the two methods were compared.